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. 2020 Apr 8;17:190–204. doi: 10.1016/j.omto.2020.03.023

Figure 4.

Figure 4

Immune Status of the Bronchoalveolar Space of Control Mice and Mice with B16-F10 Melanoma Lung Metastases after Infection with PR8 Influenza Virus

Control (dashed lines, open symbols) and tumor-carrying (lines, filled symbols) mice were infected and analyzed according to the schedule shown in Figure 3A, and the lung immune status was analyzed by flow cytometry of BALF containing cells. (A) Absolute numbers of total leucocytes (CD45.2+) as well as total numbers of macrophages (F4/80+), neutrophils (CD11b+CD11c−Ly-6G+), and T cells (CD3+) as parts of CD45.2+ cells. For principal gating strategy, see Figure S1. (B) Images represent alveolar macrophages (CD11c+CD11b−Siglec-F+) in the BALF after IAV infection. The left image shows the total number of alveolar macrophages as part of CD45.2+ cells in BALF per mouse. The middle and right images show the percentage of IL-R4α- and MHCII-positive alveolar macrophages. (C) Infiltration kinetics of the BAL space by peripheral macrophages (CD11b+CD11c−F4/80+), with the left image showing the total amount of cells per mouse and the middle and right images showing percentages of IL-R4α- and MHCII-positive cells. (D) Infiltration kinetics of the BAL space by NK cells (CD3−CD49b+), with the left image showing the total amount of NK cells per mouse and the middle and right images showing percentages of NK cells expressing the activation markers NKp46 and IFN-γ. (E and F) Infiltration kinetics of BAL space by (E) T helper (CD3+CD4+) and (F) T killer (CD3+CD8+) cells, with the left image showing the total amount of appropriate cell population per mouse and other images showing T cell populations expressing activation markers CTLA-4, PD-1, and IFN-γ. The right images of (E) show percentages of FOXP-positive Treg cells in BALF of control and tumor-containing mice at day 9 and 12 after IAV infection in comparison to their non-infected counterparts. Note that the number of NK and T killer cells in BALFs of non-infected mice was too low to obtain reliable data for cells expressing specific markers. Data are expressed as means ± SEM, with n = 4–6 animals per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Kruskal-Wallis test with Dunn’s multiple comparison test).